SUMMARY Treatment-resistant depression (TRD) affects 20-30% of major depressive disorder (MDD) patients and is defined as a failure to respond to at least two adequate rounds of treatment with standard of care antidepressants. TRD represents a substantial unmet medical need, requiring novel treatments with rapid and sustained antidepressant activity and good safety and tolerability profiles. (S)-Ketamine (Spravato, Esketamine) was recently approved for the treatment of TRD; however, it has several adverse side effects including sedation, dissociative effects, and abuse liability. The precise mechanism of action (MOA) of ketamine remains unclear; while described as a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist, it has additional non- NMDAR effects. In humans, ketamine is rapidly metabolized and some metabolites, such as (2R,6R)- hydroxynorketamine (HNK), have rapid antidepressant-like activity in rodent models. The antidepressant-like activity of HNK is partially mediated through metabotropic glutamate receptor subtype 2 (mGlu2). Thus, we propose that negative allosteric modulation of mGlu2/3 represents a promising new strategy to treat TRD. Our rationale is four-fold: (1) mGlu2/3 receptors are expressed in the forebrain regions affected in depression, with particular enrichment in the medial prefrontal cortex, hippocampus and medial habenula, (2) inhibition of mGlu2/3 leads to molecular and cellular changes similar to those caused by ketamine, including ?-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptor potentiation, brain-derived neurotrophic factor (BDNF) release, activation of mammalian target of rapamycin (mTOR) and ultimately synaptic remodeling, (3) mGlu2/3 orthosteric antagonists produce ketamine-like behavioral effects with sustained efficacy in multiple rodent procedures that model aspects of depression, and (4) the antidepressant-like activity of the ketamine metabolite (2R,6R)-HNK in rodent models is partially mediated by mGlu2. Thus, mGlu2/3 negative allosteric modulators (NAMs) are predicted to produce antidepressant effects similar to ketamine without the psychotomimetic side effects. Our objective is to advance a mGlu2/3 NAM into preclinical development and IND-enabling studies. To this end, we have identified a lead and a backup mGlu2/3 NAM with excellent drug-like properties. The specific aims are the following: (1) Determine the efficacy of our lead mGlu2/3 NAM in the rodent Probabilistic Reward Task (PRT) and Effort-related Choice (ERC) task after acute oral dosing; and (2) Determine target engagement of our lead mGlu2/3 NAM in vivo. On completion of this Phase I STTR, we will be able to determine if the lead compound and/or the backup have the efficacy and pharmacological profile to be advanced into preclinical development, which would become the basis of a Phase II application. Hence, the studies proposed here are important precedents to support a future clinical program for MDD and TRD.